Directed evolution refers to biotechnological processes for optimizing the activity of proteins by means of random changes introduced into selected respective genes. Directed evolution involves the creation of a library of mutated genes, and then selection of the mutants that encode proteins having desired properties. The process can be an iterative one in which gene products that have improvement in a desired property are subjected to further cycles of mutation and screening. Directed evolution provides a way to adapt natural proteins to work in new chemical or biological environments, and/or to elicit new functions. The potential plasticity of proteins is such that chances exist that for every new challenge, such as a new environment and desired new or altered activity, it should be possible, given a sufficient pool of modified proteins (or encoding nucleic acids), that an appropriately ‘evolved’ protein could be found that would have a desired activity. The problem is in generating and then identifying the appropriate sequence.
There have been practical approaches to this problem (see, e.g., U.S. Pat. Nos. 6,096,548; 6,117,679; 6,165,793; 6,180,406; 6,132,970; 6,171,820; 6,238,884; 6,174,673; 6,057,103; 6,001,574; 5,763,239; 5,837,500; 5,571,698; 6,156,509; 5,723,323; 5,862,514; 5,871,974; 5,779,434 and others). Typically theses approaches are of two types. One is a purely “rational” approach that is based on the assumption that the optimized proteins can be rationally designed. This, however, requires sufficient information regarding the laws that govern protein folding, molecular interactions, intra-molecular forces and other dynamics of protein activity. This rational approach is extremely dependent on a number of variables and parameters that are not known. Consequently, although useful in some specific cases and applications, the rational approach intended to ‘predict’ protein structure remains limited in applicability.
In contrast to the rational approach, random approaches have also been employed. One random approach requires synthesis of all possible protein sequences or a statistically sufficient large number of proteins and then screening them to identify proteins having the desired activity or property. Since the resources to synthesize all possible theoretical sequences of a single protein is not possible, this approach is impracticable. Other random approaches are based on gene shuffling methods, which are PCR-based methods that generate random rearrangements between two or more sequence-related genes to randomly generate variants of the gene.
The development and scope of directed evolution, thus, has been limited, and its potential remains to be exploited. In order to exploit the potential of directed evolution, alternative approaches for generating and identifying evolved proteins are needed. It is an object herein to provide methods and products to exploit the potential of directed evolution.